Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding

Abstract

Halogenation of bases is a widespread method used for solving crystal structures of nucleic acids. However, this modification may have important consequences on RNA folding and thus on the success of crystallization. We have used a combination of UV thermal melting, steady-state fluorescence, X-ray crystallography, and gel electrophoresis techniques to study the influence of uridine halogenation (bromination or iodination) on the RNA folding. The HIV-1 Dimerization Initiation Site is an RNA hairpin that can adopt an alternative duplex conformation and was used as a model. We have shown that, unexpectedly, the RNA hairpin/duplex ratio is strongly dependent not only on the presence but also on the position of halogenation.

FIGURE 1.

(A) Sequence and secondary structure of HIV-1 subtype A and subtype F DIS stem–loops used in this study. Black arrows indicate uridines substituted into C5-bromouridines, C5-iodouridines, or C5-methyluridines. (B) Secondary structure of the kissing loop–loop complex and extended duplex formed upon dimerization. (C) Schematic drawing of the molecular beacon strategy used to distinguish between hairpins from duplex forms.

FIGURE 2.

Semidenaturing polyacrylamide gel showing the proportion of extended duplex and hairpin formed at 30 μM RNA concentration in water for unmodified and several brominated subtype F DIS.

FIGURE 3.

(A) Fluorescence quantification of the relative amount of extended duplex formed in water after folding. Values obtained for unmodified and BrU3 DIS sequences are represented as circles and squares, respectively. (B) Time-dependent hairpin–duplex conversion for the unmodified and BrU3-C275 DIS sequences upon incubation with salts at 37°C. The inset shows that the duplex yield varies linearly with the logarithm of the time over three orders of magnitude. The fractions of duplex at t=0 (i.e., before salt addition) are 8% for the unmodified sequence and undetectable for the BrU3 sequence. (C) Normalized differential melting curves of C275 DIS (black), BrU3-C275 DIS (gray), and BrU2-C275 DIS (dash-dot) in salts conditions. Three UV-melting curves are superimposed in each case to assess the reproducibility of measurements in saline condition. In absence of salt (insert), the maxima of the melting curves allowed us to define reliably the difference in “melting temperature” between C275 DIS (black), BrU3-C275 DIS (gray), and T3-C275 DIS (dashed), but overall, the melting curves were much more variable (data not shown), particularly at high temperature.

FIGURE 4.

Detailed stereo views showing the potassium cation in the DIS RNA for the BrU3 at 2.0 Å resolution (A, PDB ID 2FCZ) and for the BrU2 at 1.6 Å resolution (B, PDB ID 1ZCI). The 2Fo-Fc electron density map contoured at 1.4 σ above mean level is represented in blue, and the anomalous difference map contoured at 5.0 σ above mean level is represented in red. Ligands of the potassium cation and some H-bonds are depicted with black lines.